Characterization of structure and dynamics of submicrometric systems via fluorescence correlation spectroscopy [Elektronische Ressource] / vorgelegt von Riccardo Raccis

Raccis

Sujets

• Physik

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Characterization of Structure and Dynamics
of Submicrometric Systems
via Fluorescence Correlation Spectroscopy



Dissertation



zur Erlangung des Grades

Doktor der Naturwissenschaften
im
Fachbereich Physik, Mathematik und Informatik
der Johannes Gutenberg-Universität Mainz


vorgelegt von
Riccardo Raccis
geboren in Florenz, Italien

Mainz, im Dezember 2010


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3 4





“There are more things in heaven and earth, Horatio,
Than are dreamt of in your philosophy.”
- William Shakespeare, Hamlet
5 6





To Eva, and Mariona.
I miss you girls




7 8 Summary



Summary..............................................................................................................................9
1. List of abbreviations .................................................................................................11
2. Abstract.....................................................................................................................13
2.1. English version ..................................................................................................13
2.2. German version .................................................................................................14
3. Introduction and motivation .....................................................................................17
4. FCS: general theory and practical application..........................................................21
4.1. Theory................................................................................................................21
4.2. Practical application ..........................................................................................29
5. Experimental ensemble ............................................................................................33
5.1. FCS setup...........................................................................................................33
5.2. Chambers...........................................................................................................34
5.3. Materials employed ...........................................................................................35
5.3.1. Gel constituents .........................................................................................35
5.3.1.1. PNIPAAm..........................................................................................35
5.3.1.2. BP-silane............................................................................................36
5.3.2. Solvents .....................................................................................................36
5.3.2.1. Water .................................................................................................36
5.3.2.2. Ethanol...............................................................................................36
5.3.2.3. Water-based buffers...........................................................................37
5.3.2.3.1. PBS .................................................................................................37
5.3.2.3.2. HEPES............................................................................................37
5.3.2.3.3. Acetate............................................................................................37
5.3.3. Fluorescent probes.....................................................................................38
5.3.3.1. Molecular dyes ..................................................................................38
5.3.3.1.1. Rh6G...............................................................................................38
5.3.3.1.2. Cy5 .................................................................................................38
5.3.3.1.3. Alexa 647........................................................................................38
5.3.3.2. Green Fluorescent Protein .................................................................39
5.3.3.3. Quantum dots.....................................................................................40
5.3.4. Gel functionalization material ...................................................................42
5.3.4.1. GFP antibodies ..................................................................................42
5.3.4.2. TFPS ..................................................................................................43
5.3.4.3. EDC ...................................................................................................43
5.3.4.4. Ethanolamine.....................................................................................43
5.3.5. Inverse opals..............................................................................................43
6. Experiment 1: PNIPAAm-Rhodamine interactions in a water solution..................47
6.1. Motivation .........................................................................................................47
6.2. Experimental......................................................................................................48
6.3. Diffusion and possible Models..........................................................................52
9 6.4. Data analysis and theory development..............................................................61
6.5. Conclusions .......................................................................................................96
7. Experiment 2: Size-dependent diffusion in inverse opals ........................................97
7.1. Motivation .........................................................................................................97
7.2. Experimental .....................................................................................................98
7.3. Blinking effect and FCS theory re-engineering ..............................................100
7.3.1. Data simulation........................................................................................106
7.3.2. Diffusion in iOpals and experimental-simulation comparison................111
7.4. Conclusions .....................................................................................................115
8. Experiment 3: PNIPAAm hydrogel nanostructure and dynamics for biosensing
implementation 117
8.1. Motivation .......................................................................................................117
8.2. Sample preparation..........................................................................................119
8.2.2. Gel spincoating and drying......................................................................120
8.2.3. Gel crosslinking and gel-substrate grafting.............................................121
8.2.4. Pre-FCS gel characterization and rinsing................................................122
8.2.5. Gel annealing...........................................................................................122
8.2.6. Gel functionalization ...............................................................................123
8.3. Experimental ...................................................................................................123
8.3.1. Non-annealed gels ...................................................................................126
8.3.1.1. Variable: crosslink density ..............................................................126
8.3.1.2. Variable: temperature ......................................................................133
8.3.1.3. Variable: buffer ...............................................................................141
8.3.2. Annealed gels ..........................................................................................147
8.3.2.1. Variable: protein diffusion and probe size dependence ..................151
8.3.2.2. Variable: antibody functionalization...............................................156
8.4. Conclusions .....................................................................................................165
9. Concluding remarks and outlook ...........................................................................167
10. References ..............................................................................................................171
11. Curriculum Vitae....................................................................................................177


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